Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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TITLE OF THE INVENTION
A GROUP OF ANTI-CANCER COMPOUNDS WITH SPECIFIC STRUCTURE
AND THEIR PRODUCTION METHOD
BACKGROUND OF THE INVENTION
Field of the Invention:
The invention is drawn to a group of compounds with specific structure, which
possess anti-cancer activity. It also relates to processes of making these
compounds,
and processes for their use in human and other mammalian subjects for cancer
therapy, prevention, and immune boosting functions.
Description of the Back -rgrOUnd:
Extensive studies have been underway for over 2 decades to identify drugs
that could be used to treat cancer. Drugs that either directly or indirectly
cause tumor
cell growth arrest, or cause tumor cell death via apoptotic mechanisms have
been
identified. However, major limiting factors in anticancer therapy still exist,
i.e. drugs'
adverse effects on normal cells and drug resistance developed by cancer cells.
Some
new approaches, such as anti-angiogenesis approach, represent new avenues of
fighting cancer.
Nonetheless the most direct approach to stop cancer is to use drugs that lcill
tumor cells. As mentioned above, one major drawback of these conventional
cancer-
killing drugs is that they also kill non-cancerous cells and cause severe
adverse
effects. In an effort to search for anti-cancer agents that have minimal
effects on
normal tissue, a group of compounds with specific structure that directly kill
cancer
cells but do not harm normal cells have recently been identified.
A group of specific iso- and anteiso- branched-chain fatty acids with
significant anti-cancer effect has been described in Applicant's U.S.
Application Ser.
No. 09/173,681, now U.S. patent no. Such compounds as described
in the above Applicant's U.S. Application, and derivatives thereof obtained by
reacting the acid moiety thereof, are described in Applicant's WO 99/53086.
These
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compounds have shown excellent cytotoxic activity through induction of
apoptosis
against a broad variety of cancer cells, including, but not limited to,
leukemia, breast
cancer, prostate cancer, lung cancer, with extremely low toxicity to
experimental
animals.
JP-A 04295423 and JP-B 07072134, each to Daiichi, disclose anti-cancer
agents containing MeCHR(CHZ)"COOH wherein R is Cl-CS alkyl and n=4-22. US
4,985,466 to Deguchi disclose a method for treating tumors susceptible to
treatment
with a wool fatty acid, or its reduced alcohol, metal salt or aliphatic ester
derivative,
or a wool alcohol, or its carboxylic acid, aliphatic ether or aliphatic ester
derivative.
Deguchi additionally disclose that it is characteristic of wool fatty acid and
wool
alcohol to contain a large quantity of iso- and anteiso- higher saturated
aliphatic
compounds. Deguchi also exemplifies a number of specific iso- and anteiso-
higher
saturated aliphatic acids and alcohols.
However, none of the above prior art recognizes Applicant's discovery that the
anti-cancer activity resides in a terminal branch structure and a polar group
per se
directly linked at opposite ends, respectively, of a long chain group.
SUMMARY OF THE INVENTION
The present invention relates to a group of compounds with specific chemical
structure, and possessing anti-cancer activity. This group of compounds are
comprised of three parts: end-terminal group, leading group, and a long-chain
aliphatic, non-cyclic, saturated or unsaturated, hydrocarbon group, defined in
further
detail below, to link them. The end-terminal group is isopropyl, sec.-butyl,
or te~t.-
butyl group; it must be linked by a long-chain aliphatic, non-cyclic,
saturated or
unsaturated, hydrocarbon group, to the leading group; the leading group must
be a
polar group. The more polar it is, the stronger the anti-cancer activity will
be. In
addition, this group of compounds has cancer prevention and immune boosting
functions without harmful side effects to humans and other marninalian
subjects. The
invention also relates to methods of producing these compounds. The above-
mentioned specific group of compounds can be chemically synthesized. It is
possible
that they may exist in nature, non-isolated, e.g., in living organisms.
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DETAILED DESCRIPTION OF THE INVENTION
The present invention is drawn to a specific group of compounds with unique
chemical structure. This unique structure is comprised of three parts: end-
terminal
group, leading group, and a long-chain aliphatic, non-cyclic, saturated or
unsaturated,
hydrocarbon group to link them:
I. An end-terminal group of isopropyl: CH3 ~ sec.-butyl: CH3
CH3 '
CH~
2 5
CH3
or test.-butyl: CH3 group;
CH3
2. The end-terminal group is linked to the leading group by a long-chain
aliphatic,
non-cyclic, saturated or unsaturated, hydrocarbon group, the length of which
is
at least 5 carbons, such as pentamethylene, and preferably between 5 and 19
carbons;
3. The leading group must be a polar group. The more polar this group is, the
stronger the anti-cancer activity will be. When the leading group is non-
polar,
the compound will not have anti-cancer or cancer prevention or immune boosting
functions.
This specific group of compounds can be represented by the following three
formulae (1), (2) or (3):
CH3
CH3 CH3 \
~CH(CnH2n-x~~ CH(CnHZn_~R, or CH3--~-(CnH2n-r,~R,
CH3 ~ C~H / CH3
(I) (2) (3)
wherein n is an integer of at least 5, and preferably 5 to 19, m is an even
3
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integer from 0 to 2b, wherein b is the maximum number of unsaturated bonds (a
double bond is counted as 1 and a triple bond is counted as 2), and R can be
any polar
group. However, to the extent R includes polar groups resulting in compounds
disclosed by Daiichi or Deguchi, supra, or other unknown prior art, these
compounds
are excluded herein from the compounds claimed. Thus, excluded are compounds
of
formulae (1) and (2) wherein m=0, and R is COOH or an aliphatic ester thereof
or salt
thereof, or R is OH or an aliphatic ether thereof or aliphatic ester thereof.
In the present invention, R can be, but is not limited to, the following:
1: amide group, such as R = -CONRIRZ
wherein NRl can be, but is not limited to, the following: hydroxyl-substituted
phenylamine group (wherein the number of hydroxyl groups) can be 1 to 5, and
they
can be linked to any possible position(s)); amino acid; amino-glucose; 3-
hydroxyl-4-
carboxyl-phenylamine group; sulfonic phenylamine group; or amino heterocyclic
group, while RZ is hydrogen. Alternatively, Rl and Rz can independently be
methyl or
ethyl group; or R, and RZ together with N can be a heterocyclic group, such as
piperidine, pyrrolidine, morpholine, etc.
For example,
CONH \ / OH
OH (1)
N-(3,4-Dihydroxyphenyl)-13-methyl tetradecanamide
CONH ~ ~ S03H
(2)
N-(4-Sulfo-phenyl)-13-methyl tetradecamide
CONHCH2COOH
(3)
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N-( 13-Methyl-tetradecanoyl)glycine
CH3
CONH COOH
(4)
N-( 13-Methyl-tetradecanoyl)-L-alanine
CH2
CONH CHCOOH
(5)
N-( 13-Methyl-tetradecanoyl)-L-phenylalanine
CH(CH3)2
CONH CHCOOH
(6)
N-( 13-Methyl-tetradecanoyl)-L-valine
CH~,CH(CH3)2
CONH CHCOOH
N-( 13-Methyl-tetradecanoyl)-L-leucine
CH3
CH~ CH2CH3
~--CONHCHCOOH (g)
N-( 13-Methyl-tetradecanoyl-L-isoleucine
CH2CH2COOH
~-CONH-CHCOOH
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N-(13-Methyl-tetradecanoyl)-L-glutamic acid
~- CON
COOH (10)
N-( 13-Methyl-tetradecanoyl)-L-proline
~-CONH-Glu
a
(II)
N-( 13-Methyl-tetradecyl)-2-amino-2-deoxyglucose
OH
CONH ~ COOH
(12)
N-(3-Hydroxy-4-carboxylphenyl)-13-methyl-tetradecanamide
Calls
-' '--CON\
\C2H5 (13)
N,N-Diethyl- I 3-methyl-tetradecanamide
CON
(14)
N-(13-Methyl tetradecanoyl) piperdine
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v v v v ICON
(15)
N-( 13-Methyl-tetradecanoyl) pyrrolidine
CON O
(16)
N-(13-Methyl-tetradecanoyl) morpholine
CO-NH
~N
N~
(17)
N-( 13-Methyl)-tetradecanoyl-2-amino-pyrimidine
2: ester group, such as R = -COORS
wherein R3 can be, but is not limited to, a phenyl group with substitutions)
of
sulfonic, carboxyl, or 1-5 hydroxyl group(s), at any possible position(s).
For example,
COO ~ S03H
(18)
(4-Sulfo phenyl)-13-methyl tetradecanate
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OH OH
COO O .
COON (19)
(5-Carboxy-2,3-dihydroxy phenyl)-13-methyl-tetradecanate
OH OH
COO
(20)
(2,3-Dihydroxy phenyl)-13-methyl-tetradecanate
3: ketone group, such as R = -COR4
wherein R4 can be, but is not limited to, a phenyl group with substitutions)
of
1-5 hydroxyl groups) at any possible position(s).
For example,
OH
CO O OH
(21)
(2,4-dihydroxy phenyl)-12-methyl tridecanone
OH
CO ~ OH
(22)
OH
(2,4,6-Trihydroxy phenyl)-12-methyl tridecanone
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OH OH
CO O OH
(23)
(2,3,4-Trihydroxy phenyl)-12-methyl tridecanone
4: hydroxyl-substituted benzoyloxy group, such as
(OH)n
R -O-CO
wherein the number of hydroxyl groups) can be 1 to 5, and they can be lint{ed
to any possible position(s).
For example,
OH
OCO ~ OH
OH (24)
13-Methyltetradecyl-3,4,5-trihydroxy benzoate
5: hydroxyl-substituted phenyl group, such as
(OH)n
R--
wherein the number of hydroxyl groups) can be 1 to 5, and they can be linked
to any possible position(s).
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For example,
H
OH
(25)
(13-Methyl tetradecyl)-2,4-dihydroxy phene
OH
OH
(26)
( 13,13-Dimethyl tetradecyl)-2,4-dihydroxy phene
OH
OH
(27)
(12-Methyl tetradecyl)-2,4-dihydroxy phene
OH
OH
OH (2g)
(13-Methyl tehadecyl)-2,4,6-trihydroxy phene
OH OH
OH
(29)
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(13-Methyl tetradecyl)-2,3,4-trihydroxy phene
6: amine group, such as
(1) R= -NHRS or their physiologically acceptable salt, e.g., hydrochloric.
acid
or sulfonic acid salt, etc.; wherein RS can be, but is not limited to, methyl,
ethyl,
hydroxyethyl, or a phenyl group, with substitutions) of sulfonic, carboxyl, or
1-5
hydroxyl group(s), at any possible position(s).
For example,
NH ~ S03H
(30)
N-(12-Methyl tridecyl)-4-amino benzenesulfonic acid
NH ~ COOH
(3I)
N-(12-Methyl-tridecyl)-4-amino-benzoic acid
(32)
N-(12-Methyl-tridecyl)-4-amino benzenol
NH ~ OH
OH (33)
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N-( 12-Methyl-tridecyl)-4-amino-1,2-benzenediol
H
(34)
N-(12-methyl-tridecyl) amino-acetic acid
~2) R-- -N
R7
or their physiologically acceptable salt, e.g., hydrochloric acid or sulfonic
acid
salt, etc.; wherein R6, R~ can be, but are not limited to, methyl, ethyl,
hydroxyethyl
group, etc.
For example,
.CH3
v v ~ ~ ~ ~~''/N
\CH3 35
( )
N,N-Dimethyl-12-methyl tridecyl amine
.CH2CH20H
wr w,~- ~ //N
\CH2CH20H 36
( )
N,N-Di-((3-hydroxy ethyl)12-methyl-tridecyl amine
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7: amino-acyl group, such as
R= -NH-CO-R8, wherein R8 can be, but is not limited to, a phenyl group with
substitutions) of sulfonic, carboxyl, or 1-5 hydroxyl group(s), at any
possible
position(s).
Fox example,
OH
NHCO ~ OH
OH (37)
N-(12-Methyl-tridecyl)-3,4,5-trihydroxy benzenamide
8: a-substituted carboxylic group, such as formula:
R= -CH-COOH
I
R9
where R~ can be, but is not limited to, halogen (fluorine, chlorine, bromine,
iodine), amino, hydroxyl group, etc.
For example,
COOH
38
Br ( )
2-Bromo-13-methyl-tetradecanoic acid
COOH
NHZ (39)
2-Amino-13-methyl-tetradecanoic acid
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9: heterocyclic group, such as
For example,
N,N
~~ SH
U U \/ 'w/ '~/ ~/ ~ S
(40)
2-(12-Methyl-tridecyl)-5-mercapto-1,3,4-thiadiazole
N,N
~~-OH
W./ ~./ U ~/
(41 )
2-(12-Methyl-tridecyl)-5-hydroxy-1,3,4-oxadiazole
The compounds with specific structure also include unsaturated branched-
chain fatty acids, and their pharmaceutically acceptable esters and metal
salts.
Particularly, those unsaturated branched-chain fatty acids with a double bond
between
the a and ~3 carbons from the carboxyl end have stronger anticancer activity
than other
acids with a double bond at different positions.
For example,
~COOH
(42)
15-Methyl-2-hexadecenoic acid
~COOH
(43)
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13-Methyl-2-tetradecenoic acid
The above-mentioned specifically-structured compounds can be chemically
synthesized. Chemical synthesis can be carried out according to, but not
limited to,
the following methods:
1. In the specific structure of the current invention, the aliphatic long
chain
with end-terminal isopropyl or sec.-butyl group can be obtained from
corresponding
iso- or anteiso- fatty acids, respectively; the aliphatic long chain with end
terminal
tart.-butyl group can be obtained from corresponding fatty acids with end-
terminal
tart.-butyl group. Long chain fatty acids with an end-terminal tart.-butyl
group can be
synthesized from the monoester of the corresponding long chain diacid and a
short
chain fatty acid with end-terminal tart.-butyl group. Monoester of the long
chain fatty
acid with an end-terminal tart.-butyl group can be produced following
electrolysis with
the presence of sodium methoxide in anhydrous methanol, and can be hydrolyzed
to
yield the desired fatty acid in the presence of NaOH.
The following equation shows the synthesis of 13,13-dimethyl-tetradecanoic
acid as an example:
Na, CH30H
(CH3)3CCH2COOH + HOOC(CH2)IOCOOCH3-~ (CH3)3C(CH2)11COOCH3
electrolysis
NaOH
(CH3)3C(CH2)11COOH
Example 1 has the details of the procedure.
2. In the specific structure of the current invention, when the leading group
R
is amide derivative, -CONR1R2:
Corresponding fatty acid reacts with dichlorosulfoxide to yield acyl chloride,
and the latter can react with amino acid, amino-glucose, diethylamine,
piperidine,
pyrrolidine, morpholine, amino-phenol, amino-benzoic acid, or amino-
benzenesulfonic acid to yield the desired product.
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The following equation shows the synthesis of N-(13-methyl-tetradecanoyl)-4-
amino-1,2-benzenediol [N-(3,4-Dihydroxyphenyl)-13-methyl tetradecanamide~ as
an
example:
SOC12
(CH3)2CH(CH~)COOH~ (CH3)2CH(CHa)1lCOCl
D
NH2 ~ OH
OH (CH3)2CH(CH2)11CONH O OH
OH
NaOH
Example 2 has the details of the procedure.
3. In the specific structure of the current invention, when the leading group
R
is ester derivative, -COORS:
Corresponding fatty acid reacts with dichlorosulfoxide to yield acyl chloride,
and the latter can react with hydroxy-benzoic acid, or hydroxy-benzenesulfonic
acid to
yield the desired product.
The following equation shows the synthesis of (4-sulfo phenyl)-13-methyl
tetradecanate as an example:
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SOCl2
(CH3)2CH(CH2)11COOH > (CH3)2CH(CH2)mCOCI
HO ~ S03H
(CH3)2CH(CH2)1iC00 O S03H
NaOH
4. In the specific structure of the current invention, when the leading group
R
is ketone derivative, -COR4:
Corresponding fatty acid reacts with 1,3-benzenediol to yield desired product
with the catalysis of a Lewis acid.
The following equation shows the synthesis of (2,4-dihydroxy phenyl)-12-
methyl tridecanone as an example:
HO O OH
OH
(CH3)2CH(CH2)11COOH~ (CH3)~CH(CH2)IiCO O OH
ZnCl2
5. In the specific structure of the current invention, when the leading group
R
is hydroxyl-substituted phenyl derivative:
Corresponding hydroxylphenyl aliphatic ketone can be synthesized following
method in 4 above, and can be reduced in the presence of zinc dust and
hydrochloric
acid to yield the desired product.
The following equation shows the synthesis of (13-methyl-tetradecyl)-2,4-
dihydroxy phene as an example:
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OH W H~CI OH
(CH3)2CH(CH2)uC0 O OH - (CH3)2CH(CH2)ia ~ OH
Example 7 has the details of the procedure.
6. In the specific structure of the current invention, when the leading group
R
is hydroxyl-substituted benzoyloxy derivative:
The methyl monoester of the corresponding fatty acid can be reduced to the
respective alcohol with sodium and anhydrous methanol, the latter then reacts
in an
esterification reaction with hydroxyl-substituted benzoic acid in the presence
of HCl
under reflux with toluene and water to yield the desired product.
The following equation shows the synthesis of 13-methyltetradecyl-3,4,5-
trihydroxy benzoate as an example:
Na, CH30H
(CH3)~,CH(CH2)11COOCH3 ---~ (CH3)2CH(CH~)11CH20H
OH
HOOC ~ OH OH
OH
(CH3)2CH(CH2)nCHaOCO ~ OH
HCI, toluene
OH
Example 8 has the details of the procedure.
7. In the specific structure of the current invention, when the leading group
R
is amine derivative, two kinds of methods can be applied:
(1) The methyl monoester of the corresponding fatty acid can be reduced to the
respective alcohol with sodium and anhydrous methanol; the latter can then
react with
dichlorosulfoxide to yield alkyl chloride. Alkyl chloride can react with the
amine
derivative to yield the desired product.
The following equation shows the synthesis of N-(13-methyl-tetradecyl)-4-
amino-1,2-benzenediol as an example:
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Na, CH30H SOCl2
(CH3)2CH(CH2)11COOCH3 > (CH3)2CH(CH2)nCH20H->
NH2 ~ OH
OH
(CH3)aCH(CH2)uCH2C1- ~ (CH3)2CH(CH2haNH O OH
OH
(2) Corresponding fatty acid can be converted to amide, and amide rearranges
to give amine in the presence of bromine and sodium methoxide. Amine then
reacts
with chloro-derivatives to yield the desired product.
SOC12 NH3 Br2,CH30Na
(CH3)2CH(CH2)11COOH--~ CH3CH(CH2)nCOCI > CH3CH(CH2)11CONH2 >
H2CH20H
C1CHZCH20H
(CHs)2CH(CHZ)i iNH2 -~ (CH3)2CH(CH2O tN
CHZCH20H
Example 6 has the details of the procedure.
8. In the specific structure of the current invention, when the leading group
R
is amino-acyl derivative:
These compounds can be synthesized by reaction between amine (described
above in 7) and the methyl monoester of the corresponding carboxylic acid.
The following equation shows the synthesis ofN-(12-methyl-tridecyl)-3,4,5-
trihydroxy benzenamide as an example:
OH
CH300C ~ OH
OH OH
(CH3)2CH(CH2)11NH2--~(CH3)2CH(CH2)11NHC0 O OH
OH
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9. In the specific structure of the current invention, when the leading group
R
is a-substituted carboxylic derivative:
Corresponding fatty acid can react with bromine to yield a-bromo fatty acid,
which then reacts with ammonia water to yield a-amino derivatives.
The following equation shows the synthesis of 2-amino-13-methyl-
tetradecanoic acid as an example:
Br2
(CH3)2CH(CH2)ICOOH~(CH3)ZCH(CH2)IOCHCOOH~ (CH3)2CH(CH2)IOCHCOOH
PC13 Br NH2
10. In the specific structure of the current invention, when the compounds are
unsaturated branched-chain fatty acids with a double bond between a and (3
carbons
from the carboxyl end:
The methyl monoester of the corresponding fatty acid can be reduced to the
respective alcohol with sodium and anhydrous methanol, the latter can then be
oxidized to aldehyde in the presence of a pyridine salt of chloro-chromic
acid. The
aldehyde can then be decarboxylated in a condensation reaction in the presence
of
malonic acid to yield the desired product.
The following equation shows the synthesis of 15-methyl-2-hexadecenoic acid
as an example:
Na, CH30H Cr03~ py~dine ~ HCl
(CH3)2CH(CH2)11COOCH3--> (CH3)2CH(CH2)1CH20H
CH2Cl2
CH (COOH)a
(CH3)aCH(CH2)11CH0 > (CH3)2CH(CHZ)11CH--CHCOOH
pyridine,
Example 5 has the details of the procedure.
Having generally described this invention, a further understanding can be
obtained by reference to certain specific examples which are provided herein
for
purposes of illustration only and are not intended to be limiting unless
otherwise
specified. Examples demonstrating efficacy of the compounds of the present
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invention are referred to as "Experiments;" examples demonstrating preparation
of the
compounds of the present invention are referred to as "Examples."
EXPERIMENTS
Experiment 1, In vitro anti-cancer Efficacy Comparison
Reagents: In vitro anti-cancer activities of the following compounds were
compared, which are characterized by the presently-disclosed structure and
synthesized by the presently-disclosed process. They include N-(3,4-
Dihydroxyphenyl)-13-methyl tetradecanamide, N-(4-Sulfo-phenyl)-13-methyl
tetradecamide, N-(13-methyl-tetradecanoyl) glycine, N-(13-methyl-
tetradecanoyl)-L-
alanine, N-(13-methyl-tetradecanoyl)-L-phenylalanine, N-(13-methyl
tetradecanoyl)-
L-valine, N-(13-methyl-tetradecanoyl)-L-leucine, N-(13-methyl-tetradecanoyl)-L-
isoleucine, N-(13-methyl-tetradecanoyl)-L-glutamic acid, N-(13-methyl-
tetradecanoyl)-L-proline, N-(13-methyl-tetradecanoyl)-2-amino-2-deoxyglucose,
N-
(3-Hydroxy-4-carboxyphenyl)-13-methyl-tetradecanamide, N,N-Diethyl-13-methyl-
tetradecanamide, N-(13-Methyl tetradecanoyl) piperidine, N-(13-Methyl-
tetradecanoyl) pyrrolidine, N-(13-Methyl-tetradecanoyl) morpholine, N-(13-
Methyl-
tetradecanoyl)-2-amino-pyrimidine, (2,4,-dihydroxy phenyl)-12-methyl
tridecanone,
(2,4,6-Trihydroxy phenyl)-12-methyl tridecanone, (2,3,4-Trihydroxy phenyl)-12-
methyl tridecanone, (13-Methyl tetradecyl)-2,4-dihydroxy phene, (13,13-
Dimethyl
tetradecyl)-2,4-dihydroxy phene, (12-Methyl tetradecyl)-2,4-dihydroxy phene,
(13-
Methyl tetradecyl)-2,3,4-trihydroxy phene, N-(12-Methyl tridecyl)-4-amino
benzenesulfonic acid, N-(12-Methyl-tridecyl)-4-amino-1,2-benzenediol, 2-Bromo-
13-
methyl-tetradecanoic acid, 2-Amino-13-methyl-tetradecanoic acid, 15-Methyl-2-
hexadecenoic acid, 2-(12-Methyl-tridecyl)-5-mercapto-1,3,4-thiadiazole, 13,13-
Dimethyl-tetradecanoic acid, N-(13-Methyl tetradecyl)-4-amino-1,2-benzenediol,
and
9-Methyl-2-decenoic acid.
The above compounds were dissolved in DMSO at stock concentration of 10
mg/ml and stored at 4°C. Before each experiment they were diluted to
desired
concentration with serum-free RPMI 1640 medium.
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Methods:
1. K562 human leukemia cell line: K562 cells were maintained in RPMI 1640
medium with 5% fetal bovine serum at 37°C under 5% COZ atmosphere.
4x104
cells/well were plated on 96-well plate. After treatment with the test
compounds and
solvent control (DMSO) for 48 hours, numbers of live cells were counted by
trypan
blue dye exclusion. ICso and IC9o were calculated by Bliss method and listed
in Table
1.
2. B 16 mouse melanoma cell line: B 16 cells were maintained under the same
condition as above. 2x104 cells/well were plated on 48-well plate and left for
12 hours
for attachment. Cells were then treated with the test compounds and solvent
control
for 48 hours. Numbers of live cells were determined by SRB method. Briefly,
cells
were fixed with SO,uI/well 15% TCA fox 1 hour, and then washed three times
with
double-distilled water. After drying SO,uI/well 1% SRB dye was added and
reacted for
minutes. Plate was washed with 1% acetic acid. 0.2 ml Tris buffer (pH 9.6) was
added and ODs~o was recorded. ICSO and IC9o were calculated and listed in
Table 1.
Table 1, ICso and IC9o (,ug/ml) for K562 and B 16
Compounds K5 62 B 16
IC9o ICso IC9o ICso
1 N-(3,4-Dihydroxyphenyl)-13-10.1 4.98 9.28 5.12
methyl tetradecanamide
2 N-(4-Sulfo-phenyl)-13-methyl37.8 27.1 35.7 25.4
tetradecamide
3 N-(13-methyl-tetradecanoyl)21.9 14.7 20.1 12.8
glycine
4 N-(13-methyl-tetradecanoyl)-L-20.7 11.2 18.4 14.8
alanine
5 N-(13-methyl-tetradecanoyl)-L-19.6 10.3 20.5 13.4
phenylalanine
6 N-(13-methyl-tetradecanoyl)-L-22.5 18.1 19.2 11.7
valine
7 N-(13-methyl-tetradecanoyl)-L-30.6 11.8 20.1 10.4
leucine
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Compounds KS 62 B 16
8 N-(13-methyl-tetradecanoyl)-L-28.6 19.3 18.9 11.1
isoleucine
9 N-(13-methyl-tetradecanoyl)-L-37.9 28.2 35.1 26.3
glutamic acid
N-(13-methyl-tetradecanoyl)-L-25.3 17.2 17.3 15.2
proline
11 N-(13-methyl-tetradecanoyl)-2-38.1 24.3 34.6 24.5
amino -2-deoxyglucose
12 N-(3-Hydroxy-4- 24.7 18.7 22.9 16.5
carboxylphenyl)-13-methyl-
tetradecanamide
13 N,N-Diethyl-13-methyl- 42.4 9.10 31.9 19.3
tetradecanamide
14 N-(13-Methyl tetradecanoyl)38.5 18.4 33.7 17.2
piperidine
N-(13-Methyl-tetradecanoyl)25.0 16.2 17.3 13.1
pyrrolidine
16 N-(13-Methyl-tetradecanoyl)20.4 15.9 19.2 11.9
morpholine
17 N-(13-Methyl-tetradecanoyl)-2-19.2 14.8 18.6 12.3
amino-pyrimidine
18 (2,4,-dihydroxy phenyl)-12-8.90 6.42 8.12 5.91
methyl tridecanone
19 (2,4,6-Trihydroxy phenyl)-12-6.80 5.13 6.11 4.93
methyl tridecanone
24 (2,3,4-Trihydroxy phenyl)-12-20.2 15.3 19.32 14.8
methyl tridecanone
21 (13-Methyl tetradecyl)-2,4-5.25 3.62 5.14 3.22
dihydroxy phene
22 (13,13-Dimethyl tetradecyl)-2,4-5.36 3.92 5.42 3.63
dihydroxy phene
23 (12-Methyl tetradecyl)-2,4-14.6 9.51 13.8 8.81
dihydroxy phene
24 (13-Methyl tetradecyl)-2,3,4-19.2 13.6 19.5 12.8
trihydroxy phene
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Compounds K562 B 16
25 N-(12-Methyl tridecyl)-4-amino20.0 15.1 18.8 13.5
benzenesulfonic acid
26 N-(12-Methyl-tridecyl)-4-amino-13.5 6.73 12.6 5.81
1,2-benzenediol
27 2-Bromo-13-methyl-tetradecanoic10.1 7.21 11.2 6.10
acid
28 2-Amino-13-methyl-tetradecanoic28.6 19.7 25.3 17.4
acid
29 15-Methyl-2-hexadecanoic 10.1 17.7 6.8 10.5
acid
30 2-(12-Methyl-tridecyl)-5-8.75 6.10 8.05 5.16
mercapto-1,3,4-thiadazole
31 13,13-Dimethyl-tetradecanoic37.5 24.6 35.6 20.4
acid
32 N-(13-Methyl tetradecyl)-4-11.5 6.31 10.6 5.75
amino-1,2-benzenediol
33 9-Methyl-2-decenoic acid 26.7 51.2 40.5 91.0
In contrast, the specific structured compounds disclosed in the present
invention are not toxic against normal human peripheral blood lymphocytes at a
concentration above 400,ug/ml, while they have significant cytotoxicity on
various
human cancer cell lines at well below this concentration.
Furthermore the i~ vitro anti-cancer activity of 2-(12-Methyl-tridecyl)-5-
mercapto-1,3,4-thiadiazole has been tested on other human cancer cell lines,
including
human prostate cancer DU145, human gastric cancer SNU-1, human lung cancer
H1688, human colon cancer HCT-116, human hepatocaxcinoma SNU-423, human
pancreatic cancer CRL-1687, human CNS cancer SF-268, human renal cancer Caki-
1,
and human melanoma SK-MEL-5. Using viability assessment by trypan blue dye
exclusion and calculation of inhibition rate with linear regression, IC9o of 2-
(12-
Methyl-tridecyl)-5-mercapto-1,3,4-thiadiazole on various cancer cell lines
above were
determined to range from 8.3,ug/ml to 23.2~cg/m1.
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Experiment 2 Ih vitro anti-cancer Efficacy Comparison f21
The in vitro efficacy test was carried out to compare unsaturated branched
chain fatty acid, 9-methyl-2-decenoic acid and 9-methyl-6-decenoic acid, as
well as
15-methyl-2-hexadecenoic acid and 15-methyl-6-hexadecenoic acid. The results
are
shown in Table 2.
Reagents and Methods: same as in Experiment 1.
Table 2, Ih vitro anticancer activity comparison of
unsaturated fatty acids
Compound K562 B 16
ICso IC9o ICso IC9o
9-methyl-decanoic acid 55.5 87.0 97.0 280
9-methyl-6-decenoic acid 50.8 83.0 90.0 269
9-methyl-2-decenoic acid 26.7 51.2 40.5 91.0
15-methyl-hexadecanoic 25.9 35.6 20.7 38.2
acid
15-methyl-6-hexadecenoic 23.8 30.5 21.3 35.2
acid
15-methyl-2-hexadecenoic 10.1 17.7 6.8 10.5
acid
The above result suggests that the anticancer activity of unsaturated branched-
chain fatty acid is stronger than that of its corresponding saturated one, and
also for
unsaturated branched-chain fatty acids, the closer the double bond is to the
carboxyl
end, the higher polarity the molecular has, and the greater the anti-cancer
activity it
has.
Experiment 3 In vivo anti-cancer Efficac~Comparison (solid tumor model)
Mouse cervical carcinoma UI4 model was used to compare in vivo anti-cancer
efficacy of 15-methyl-2-hexadecenoic acid, 13-methyl-tetradecanoic acid and
the
chemotherapy drug cytoxan (CTX).
Reagents and Methods:
40 male ICR strain mice, weighing 19-22g, were randomly divided into 4
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groups of 10 each. U14 cervical carcinoma mass (about 2 mm3 each) was
transplanted
subcutaneously into the right armpits of all animals following standard
procedure.
The test group was given 15-methyl-2-hexadecenoic acid and 13-methyl-
tetradecanoic
acid at 0.4 glkg, respectively, intragastrically (i.g.) daily for 11 days. The
positive
control group was given a single dose of CTX i.g. (25 mg/kg) on day 1.
Negative
control group was given normal saline (N.S.) at 0.2m1/lOg daily for 11 days.
On the
12th day, all mice were sacrificed and the tumors were isolated and weighed.
The
tumor inhibition rate (TIR) was calculated and subject to t test. A comparison
of the
in vivo efficacy is shown in Table 3.
Table 3, In vivo Efficacy Comparison
Group Dosage No. Body Tumor TIR p
W. (g, W.
X~SD)
(g, LSD)
Di Diz
N.S. 20m1/kg 10 20.60.7 24.80.8 2.630.29-- --
13-methyl- 0.4g/kg 10 20.70.7 26.02.6 0.930.3264.6 <0.05
tetradecanoic
acid
15-methyl-2-0.4g/kg 10 20.60.7 25.50.8 0.500.2981.0 <0.05
hexadecenoic
CTX 25mg/kg 10 20.60.5 22.61.2 0.460.1782.5 <0.05
The above results indicate that 15-methyl-2-hexadecenoic acid has comparable
anticancer effect with CTX, and greater anticancer activity than that of
saturated fatty
acid. Judged by changes in body weights, no toxic effect was observed in
animals
treated with the test compounds of the present invention.
Experiment 4 In vivo anti-cancer Efficac,~Comparison~ascites modell
Reagents and Methods:
30 female ICR strain mice, weighing 18-21 g, were randomly divided into 3
groups of 10 each. 0.2 ml of HAC cell suspension (10' cells/ml) was injected
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intraperitoneally (i.p.) into all animals. The test group was given 2-(12-
Methyl-
tridecyl)-5-mercapto-1,3,4-thiadiazole at 0.4 g/kg i.g. daily till death. The
positive
control group was given 5-Flurouracil (5-Fu) i.g. at 25 mg/kg on day 1, 3, and
5. The
negative control group was given N.S. at 20 ml/kg daily as in test groups. The
length
of survival of each group was recorded and rate of extension of life span was
calculated and subject to t test.
Results:
Table 4. Extension of life span of HAC mice by the sample
Group No. Body Weight Survival Extension
of (g~ ~~SD) Time Rate
(%)
mice (days,
before after X~SD)
treatmenttreatment
N.S.20m1/kg 10 19.51.2 34.63.8 9.71.9 --
(Control)
2-(12-Methyl-tridecyl)-10 19.61.3 46.56.1 16.71.8 72.2
5-mercapto-1,3,4-
thiadiazole, 0.4g/kg
5-FU 25mg/kg 10 19.51.2 43.412.814.31.5 47.4
(Positive Control)
Experiment 5 Therapeutic Effect of 13-methyl-2-tetradecenoic acid on Human
Breast Carcinoma MCF7ras Xenografted into Nude Mice
Reagents and Methods:
Thirty female Balb/c-nu/nu athymic mice, 6 weeks old, weighing 18-22g, were
housed in specific pathogen free (SPF) condition throughout the course of the
experiment.
Subcutaneous transplantation of human breast carcinoma MCF7ras was carried
out under aseptic condition. Tumor mass was cut into fragments (approximately
0.18
x 0.18 x 0.20 mm in size), and injected s.c. into the right mammary fat pad of
each
animal with a trocar. The mice were randomized into control and test groups of
ten
mice each. The day after transplantation, the mice in the test group were
given
through gavage 13-methyl-2-tetradecenoic acid at 400 mg/lcg daily. The mice in
two
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control groups were administered with CTX at 25mglkg and N.S. at 0.2m1110g.
All
animals were sacrificed on day 23. The body weights and tumor weights were
measured. The tumor inhibition rates (TIR) were determined by comparing the
mean
tumor weight of the test groups (T) with that of the control group (C) and
expressed as
a (C - T)/C percentage, and were analyzed by Student's test for statistical
significance.
The results are shown in Table 5.
Table 5, Comparison of inhibition effects on transplanted
human breast cancer MCF7ras
Group No. Body weight Tumor TIR p
(g, X~SD) W.
(g, X~SD)
Di Dzs
N.SØ2m1110g 10 20.20.6 21.51.3 1.830.53-- --
CTX 25mg/kg 10 20.40.4 18.60.2 0.430.1576.5 <0.05
13-methyl-2- 10 20.30.5 24.41.1 0.470.3174.3 <0.05
tetradecenoic
acid
The results above showed significant inhibition effect of 13-methyl-2-
tetradecenoic acid on human breast cancer MCF7ras while no toxic effect was
observed from the body weight curve.
Experiment 6 13. 13-Dimeth~-tetradecanoic acid's function in preventing
ultraviolet B ra.~(UVBI-induced skin cancer
13, 13-Dimethyl-tetradecanoic acid (Sample) was encapsulated with liposome
at a final concentration of 10 %.
Thirty female SKH-1 hairless mice were randomly divided into control and test
groups of 15 each. Each mouse in both groups was treated topically once with
DMBA
(5.12 ~,g dissolved in 200 ~l acetone solution) to achieve tumor initiation.
One week
later (day 8), animals in test group started to receive topical application of
200 ~,1
Sample solution once a day. Control group received 200 ~l liposome solvent
instead
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every day. Thirty minutes after the application, animals in both groups were
exposed
to UVB (290-320 mn) radiation at the dosage of 180 mJ/cmz per day to induce
growth
of tumor. The animals were evaluated for tumor development at the end of 30
weeks.
The results suggest that the Sample has a preventive effect when used during
early stage of tumor induction. At the end of experiment, the animals in
Sample-
treated group showed a 65% reduction in tumor incidence compared to those in
the
control group. The average size of cancer in the Sample-treated group was also
87%
smaller.
The compounds with the specific structure disclosed in present invention,
including, but not limited to, 13, 13-Dimethyl-tetradecanoic acid, have cancer
prevention function, not limited to skin cancer. Since these compounds have
extremely low toxicity, they should be able to protect human and animal from
cancer
if taken regularly.
Experiment 7 Influence ofN-(13-meth,~~l-tetradecano~~4-amino-1~2-
benzenediol on Immune Functions
1: Phagocytic function of reticuloendothelial system
50 female ICR strain mice, weighing 19-24g, were randomly divided into 5
groups of 10 each. One group was given N.S. at 20 ml/kg i.g. as a normal
control. A
positive control group was given CTX i.g. at 25 mg/kg on day 1 and 6. A third
group
was given N-(13-methyl-tetradecanoyl)-4-amino-1,2-benzenediol (Sample) only at
400mg/kg i. g. The remaining two groups were given both CTX (25 mg/kg on day 1
and 6) and Sar~aple (100mg/kg and 400mg/kg, respectively). All treatments
except
CTX were administered daily for 9 days. 30 minutes after the last
administration,
O.15m1 Yidege (1:10) was injected into the tail vein of each mouse. 1 minute
and 5
minutes after, blood was drawn and 20 ~,1 serum was mixed with 0.1% NazC03.
OD6go
was measured and the clearance index K = (IgOD~- lgOD2)/(t2 t1) was
calculated. The
results were subject to t test and shown in Table 6.
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Table 6. Effects of Sample on Serum Clearance Index of Carbon Grain
in Normal and Immune-compromised Mouse
Group (i.g.) No. of K (X~SD) P
Mice
Compared Compared with
with Group Group 2
1
N.SØ2m1/lOg 10 0.07820.0342 <0.001
CTX 25mg/kg 10 0.02380.0161<0.001
Sample 400mg/kg10 0.08120.0290>0.05 <0.001
Sample 100mg/kg10 0.04120.0310<0.001 <0.001
+ CTX 25mg/kg
Sample 400mg/kg10 0.05430.0410<0.001 <0.001
+ CTX 25mg/kg
The results in Table 6 demonstrate that N-(13-methyl-tetradecanoyl)-4- amino-
1,2-benzenediol had no obvious effect on the Clearance Index of Carbon grain
in
normal mouse (P1,3 >0.05). Moreover the Sample could even improve the
clearance of
Carbon grain in CTX-treated mouse to a certain extent (both Pz,4 and PZ,S
<0.01).
2: Serum hemolysinogenesis
50 male ICR strain mice, weighing 20-23g, were randomly divided into 5
groups of 10 each. The method of administration in each group was the same as
above
(phagocytic function). However, on the 6th day after administration, 0.2 ml
3:5 (VN)
sheep red blood cell (RBC) suspension was injected i.p. into each mouse. 4
days later
(day 10), blood was drawn from all animals and serum was prepared and then
diluted
600 times. l ml diluted serum was mixed with 0.5 ml 10% sheep RBC suspension.
N.S.
was used as blank control. All samples were incubated at 37°C for 30
minutes, and
centrifuged (2000 rev/min) for 5 minutes. The supernatant was collected for
measurement of ODsao and HCso was calculated. The results were subject to t
test and
are shown in Table 7.
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Table 7. Effects of Sample on Serum Hemolysinogenesis
in Normal and Immune-compromised Mouse
Group (i.g.) No. of HCSO (X~SD)P
Mice
Compared Compared with
with Group 2
Group 1
N.SØ2m1/lOg10 33.91+2.23 <0.001
CTX 25mg/kg 10 22.514.30 <0.001
Sample 0.4g/kg10 31.484.60 >0.05 <0.001
Sample O.lg/kg10 19.50+3.20 <0.001 >0.05
+ CTX 25mg/kg
Sample 0.4g/kg10 23.413.75 <0.001 >0.05
+ CTX 25mg/kg
The results in Table 7 show that N-(13-methyl-tetradecanoyl)-4-amino-1,2-
benzenediol had no obvious effects on serum hemolysinogenesis in either normal
or
immune-compromised mouse (P1,3, Pz,4, and P2,5 >0.05). This suggests that the
Sample
does not evidently affect host humoral immunity.
3: Delayed hypersensitivity
50 female ICR strain mice, weighing 18-21g, were randomly divided into 5
groups of 10 each. The method of administration in each group was the same as
above
(phagocytic function). However, on the Sth day after administration, each
mouse was
injected with 50 ~,1 10% sheep RBC suspension through tail vein. 4 days later
(day 9),
each mouse was injected intradermally with 20 w1 10 sheep RBCsuspension or
normal
saline into metatarus of the left or right hind limbs, respectively. The
digital thickness
of each mouse was measured 24 hours later, and the difference in thickness
between
left and right digits was subject to t test analysis. The results are shown in
Table 8.
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r
Table 8. Effects of Sample on delayed hypersensitivity of
normal and immune-compromised mice
Group (i.g.) No. Difference P
of in
Mice thickness
between left
and
right palm Compared Compared to
(mm to
(X~SD) Group one Group two
N.SØ2m1/lOg 10 2.040.50 <0.01
CTX 25mg/kg 10 1.25+0.48 <0.01
Sample 0.4g/kg10 1.980.61 >0.05 <0.01
Sample O.lg/kg10 1.560.22 >0.05 >0.05
+ CTX 25mg/kg
Sample 0.4g/kg10 1.50+0.48 >0.05 >0.05
~
+ CTX 25mg/kg
Table 8 shows that N-(13-methyl-tetradecanoyl)-4-amino-1,2-benzenediol had
no inhibitory effect to the delayed hypersensitivity of both normal and CTX-
treated
mice. The values of P1,3, P2,4, and P2,5 were all greater than 0.05,
suggesting that there
were no apparent effects of the sample on host cellular immunity.
4: Weights of immune organs
The mice were sacrificed after delayed hypersensitivity test above and the
thymus and pancreas were collected and weighed. Indices of each organ (mg / l
Og
body weight) were calculated and subject to t test, and shown in Table 9 as
well.
Table 9. Effect of the Sample on the indices of immune organs of
both normal and CTX-compromised mice
Drug (i.g.) Number Indices of thymus Indices of
of
mice (mg/lOg avoirdupois)pancreas (mg/lOg
avoirdupois)
N.S. 0.2 ml/lOg10 3.623~1.369a 4.513~0.895a
CTX 25 mg/kg 10 1.3870.701 2.2871.023
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Sample 0.4 g/kg10 3.505~1.212a 4.132~0.487a
Sample 0.1 g/kg10 2.591~0.517a 2.3950.894
+
CTX 25 mg/kg
Sample 0.4 g/kg10 3.165~0.635a 3.0851.152
+
CTX 25 mg/kg
aP<0.001, compared to the control group.
The results in Table 9 show that there was no significant effect of the Sample
on the indices of immune organs for normal mice (both thymus and pancreases,
P,,3>0.05). As to the mice that were treated by CTX, these indices increased
after
combined administration of the Sample. The increase in the indices of thymus
among
these three groups is statistically significant (P1,2 and P2,ø<0.001). It is
suggested that
the Sample is different from the anticancer compounds of common clinical use,
in that
the Sample does not inhibit host immune function at therapeutic dosage.
Similarly, it was shown that the specific structured compounds disclosed in
the
present invention have no influence on immune function of normal body and
would
not aggravate the immune suppression induced by chemotherapy drugs.
Furthermore
they have immune boosting effects and alleviate the immune suppression when
used in
combination with other chemotherapy drugs.
Experiment 8 Acute Toxicity Tests of 15-methyl-2-hexadecenoic acid on
Mice
The acute toxicity tests were assessed for two administration routes, through
gavage and subcutaneous injection.
1: Gavage Administration
A preliminary test failed to establish LDSO of 15-methyl-2-hexadecenoic acid
(Sample). Thus, the acute toxicity test was conducted in twenty ICR strain
mice,
weighing 18-20g. The Sample was given through gavage at dosage of 2.Sg/kg,
twice a
day. Continuous monitoring was made for 14 days. No major anomaly was
observed,
indicating safety of the Sample with LDSO>5 g/kg.
2: Subcutaneous Injection
Twenty ICR strain mice, weighing 18-20g were used in the test. The daily
dosage of the Sample at 1 g/kg was subcutaneous injected at both sides of
spine for 14
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days. No mice death was observed, indicating safety of the Sample and
LDSO>2g/kg
(s.c.).
Experiment 9 Mechanism of anti-cancer activity of 15-methyl-2-hexadecenoic
acid
1: 15-methyl-2-hexadecenoic acid (the Sample) causes apoptosis
The test Sample kills cancer cells by induction of apoptosis in these cells.
This
is demonstrated in the following two experiments:
(1) in situ cell death detection
Leukemia K562 cells and gastric carcinoma SNU-423 cells were treated with
the Sample and slides were prepared with cell suspension. The slides were
examined
with in situ cell death detection kit (Boehringer Mannheim). Large portion of
cancer
cells after treatment of 4 hours and 8 hours were stained, suggesting that the
Sample
induced apoptosis in these cells.
(2) DNA fragmentation assay
After treatment of the Sample in the above two cell lines, cellular DNA was
purified and tested with ApoAlert LM-PCR kit (Clontech). Samples collected at
8
hours and 24 hours past treatment showed fragmented DNA in gel
electrophoresis,
which confirmed the apoptosis induced by the Sample.
2: apoptotic induction by 15-methyl-2-hexadecenoic acid is closely related to
the caspase pathway
The mechanism by which the Sample induces apoptosis is closely related to the
caspase pathway. This is suggested by the following:
(1) translocation of cytochrome c from mitochondria to cytosol
After treatment with the Sample, as early as 4 hours, cytochrome c can be
detected in the cytosol by western analysis. This suggested that cytochrome c
translocated from mitochondria to cytosol, which has been repeatedly
implicated as an upstream event of caspase activation.
(2) cleavage of many substrates of caspase, including Lamin-B, Rb, and PARP
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By western analysis, cleavage products of Lamin-B, Rb, and PARP can be
detected as early as 4 hours after treatment by the Sample. Presence of these
cleavage products pointed to caspase activity, as many other reports had
suggested. Dephosphorylation of Rb was also observed, in accordance with
the report of induction of apoptosis in segregated cancer cells by
dephosphorylated Rb.
The compounds with the specific structure in the present invention, whether
chemically synthesized or extracted from natural resources, or administered in
a
natural mixture without extraction, have significant anti-cancer activity for
human or
animal. These compounds can also be taken orally or by injection, in the forms
of
liquid, powder, tablet, injection, capsule, or encapsulated liposome, or they
can be
topically applied in the forms of cream, ointment, or lotion.
EXAMPLES
Example 1
Synthesis of 13,13-dimethyl-tetradecanoic acid
Electrolysis reaction was carried out with methyl dodecandioic monoester (139
g, O.S6 mole), 1,2-dimethyl propanoic acid (S6 g, O.S6 mole), sodium (0.65 g,
0.028
mole), and anhydrous methanol (1390 ml) at a direct current of 2.0 A until the
reaction
mixture was basic. Methanol was removed by evaporation; the mixture was washed
with water and subject to vacuum fractional distillation.
120°C/0.4mrnFIg fraction was
collected as 13,13-dimethyl-tetradecanoate (21.5 g). 13,13-dimethyl-
tetradecanoate
was then refluxed with 10% NaOH (S3.S ml) and methanol (64.5 ml) for 2 hours,
and
methanol is again removed by evaporation. The remainder was adjusted to pH 2.0
with 10% HCI, cooled down, and precipitates were collected by filtration.
Filtrate was
washed to neutral with water (became white solid) and subject to vacuum
fractional
distillation. 170°C/0.4mmHg fraction was collected and recrystallized
with ethanol-
water mixture to yield final product, 13,13-dimethyl-tetradecanoic acid (15.8
g, m.p.
37-39°C).
3S
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Example 2
Synthesis ofN-(3, 4-dihydroxyl-phenyl)-13-methyl-tetradecanamide
p-nitrophenol (69.55 g, 0.5 mole) was dissolved in 10 N HCl (200 ml) in a
1000 ml flask with heating and then cooled down. To the mixture was added 600
ml
potassium chlorate (61.25 g, 0.5 mole) solution, gradually with stirring.
Stirring was
continued for 1 hour after completion of addition and mixture was left
overnight at
room temperature. After filtration with vacuuming, the filtrate was washed
with
distilled water and recrystallized with dilute acetic acid solution to yield
pale yellow
acicular crystal, 2-chloro-4-nitro-phenol (80.28, m.p. 110-111°C, yield
rate 91%).
2-chloro-4-nitro-phenol (64.44 g, 0.4 mole) and 300 ml 4M NaOH solution
were refluxed with heating for 6 hours. After cooling down and filtration with
vacuuming, the filtrate (crystal) was dissolved in a little warm water
(60°C). The
solution was adjusted to pH 2.0 with 10% HCl and cooled down while stirring.
After
filtration, the filtrate was recrystallized with water to yield 4-nitro-1,2-
benzenediol
(52.1 g, yield rate 84%).
To a 1000 ml flask was added 4-nitro-1,2-benzenediol (49.6 g, 0.32 mole), and
then 403 ml water and 16 ml 10 N HCl (0.16 mole). While stirring 83.2 g zinc
dust
(1.28 mole) was added gradually and reaction mixture refluxed with heating for
8
hours. After cooling down and filtration with vacuuming, the filtrate was
washed
thoroughly with 95% ethanol and decolorized by active carbon. Filtration again
and
evaporation of ethanol was carried out until the solution just started to
become turbid.
After cooling in freezer and filtration with vacuuming, filtrate was collected
as 4-
amino-1,2-benzenediol (30.0g, yield rate 75%).
Dissolve 4-amino-1,2-benzenediol (23.2 g, 0.18 mole) in 95% ethanol. Under
salt ice bath 140 ml 2M NaOH solution and 150 ml dichloromethane solution of
13-
methyltetradecanoyl chloride (23.2 g, 0.18 mole) were added simultaneously to
the
benzenediol solution. Stir at room temperature for 6 hours. Adjust pH to 3.0
with
HCI, freeze, and filtrate. Wash the filtrate to neutral with water and
recrystallize with
50% ethanol to yield N-(3, 4-dihydroxyl-phenyl)-13-methyl-tetradecanamide
(55.8 g,
m.p. 85-86°C, yield rate 85%).
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Example 3
1. Synthesis ofN-(13-methyl)-tetradecanoyl glycine
13-methyltetradecanoic acid (600 mg, 2.48 mmole) and dichlorosulfoxide (2.0
ml) were added to a 10 ml round bottom flask and heated with reflux for 4
hours. To
this solution was added 10 ml petroleum ether to remove dichlorosulfoxide. The
product (13-methyl-tetradecanoyl chloride) was used in following reaction.
Glycine (186.1 mg, 2.48 mmole) and 2M NaOH (2.48 ml, 4.96 mmole) were
added to a 50 ml flask and stirred to complete dissolution. To this solution,
2M NaOH
(2.48 ml, 4.96 mmole) and above acyl chloride product in 10.0 ml of
dichloromethane
were added simultaneously by two funnels under ice bath. After complete
addition the
ice bath was removed and the reaction mixture was stirred for 6 hours at room
temperature. Large amount of white precipitates should form in the reaction
mixture
at this step. Ethanol was added to dissolve the precipitates and the organic
solvent was
removed by evaporation. Small amount of water was added and the reaction
mixture
became clear. After adding 10% dilute HCl by drops, white precipitates formed
again
and were collected by filtration. The filtrate was purified by
recrystallization with
ethanol-water mixture to yield final white solid product, N-(13-methyl)-
tetradecanoyl
glycine (0.52 g, m.p. 78-81 °C, yield rate 70.1 %). IH-NMR(CDC 13):8
5.98(m, 1H,
NH), 4.09(m, 2H, CHz), 2.27 (m, 2H, COCHZ), 1.62(m, 2H, CHZ), 1.50(m, 1H, CH),
1.15-1.32(m, 18H), 0.86(d, 6H, 2CH3); MS(m/e): 299(M+, 2), 130(25), 117(100),
99(60), 76(30), 44(30).
2. Synthesis of N,N-diethyl-13-methyl-tetradecanamide
13-methyltetradecanoic acid (726 mg, 3.0 mmole) and dichlorosulfoxide (2.0
ml) were added to a 10 ml round bottom flask and heated with reflux for 4
hours. To
this solution was added 10 ml petroleum ether to remove dichlorosulfoxide. The
product (13 methyl-tetradecanoyl chloride) was used in following reaction.
Diethylamine (548 mg, 7.5 mmole), N,N-dimethylaminopyridine (DMAP) (30
mg) and dichloromethane (5 ml) were added to a 50 ml flask and stirred to
complete
dissolution. To this solution, above acyl chloride product in 10.0 ml of
dichloromethane were added under ice bath. After complete addition the ice
bath was
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removed and the reaction mixture was stirred for 8 hours at room temperature.
10 ml
10% dilute HCl was then added and reaction stirred for 30 minutes. 20 ml water
was
added, followed by extraction with ethyl acetate (EtOAc) (30 inl x3). The
combined
organic layer was washed with water to neutral and dried with anhydrous
magnesium
sulfate. After concentration, pale yellow oily liquid, N,N-diethyl-13-methyl
tetradecanamide (0.73 g, yield rate 81.9%) was obtained. 1H-NMR(CDCl3): 8
3.34(m,
4H, 2NCH2), 2.85(t, 2H, COCHZ), 1.62(m, 2H, CHZ),1.49(m, 1H, CH), 1.07-1.26(m,
24H), 0.86(d, 6H, 2CH3); MS(m/e): 297(M+, 2), 128(25), 115(100), 100(25),
58(35),
43(20).
3. Synthesis of N-(13-methyl)-tetradecanoyl piperidine
13-methyltetradecanoic acid (1.0 g, 4.1 mmole) and dichlorosulfoxide (2.0 ml)
were added to a 10 ml round bottom flask and heated with reflux for 4 hours.
To this
solution was added 10 ml petroleum ether to remove dichlorosulfoxide. The
product
(13-methyltetradecanoyl chloride) was used in the following reaction.
Piperidine (850.6 mg, 10 mmole), N,N-dimethylaminopyridine (DMAP) (30
mg) and dichloromethane (5 ml) were added to a 50 ml flask and stirred to
complete
dissolution. To this solution, above acyl chloride product in 10.0 ml of
dichloromethane were added under ice bath. After complete addition the ice
bath was
removed and the reaction mixture was stirred for 8 hours at room temperature.
I O ml
10% dilute HCl was then added and reaction stirred for 30 minutes. 20 ml water
was
added, followed by extraction with ethyl acetate (EtOAc) (30 m1 x3). The
combined
organic layer was washed with water to neutral and dried with anhydrous
magnesium
sulfate. After concentration, pale yellow oily liquid, N-(13-methyl)-
tetradecanoyl
piperidine (1.01 g, yield rate 79.7%) was obtained. The final product will
solidify
when placed in refrigerator. m.p.< 0°C. 1HNMR(CDCl3): 8 3.52(brs, 2H,
NCHZ),
3.38(brs, 2H, NCHZ), 2.29(t, 2H, COCHZ), 1.44-1.62(m, 9H), 1.12-1.28(m, I8H),
0.83(d, 6H, 2CH3); MS(m/e): 309(M+, 2), 294(10), 140(70), 127(100), 112(25),
84(40), 43(35).
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Example 4
Synthesis of (3, 4-dihydroxyphenyl)-13-methyl-tetradecanamine
Dichlorosulfoxide (143.5 ml, 2.0 mole) was added to 13-methyl-1-tetradecanol
(114.0 g, 0.5 mole) and heated with reflux for 4 hours. To this solution was
added 200
ml petroleum ether to remove dichlorosulfoxide. After vacuum fractional
distillation,
159-161°C/l.OmmHg fraction was collected as 13-methyl-1-chloro-
tetradecane (102 g,
yield rate 82.8%).
4-amino-I,2-benzenediol (25 g, 0.2 mole) was dissolved in 200 ml methanol.
To this solution, I3-methyl-I-chloro-tetradecane (54.2 g, 0.22 mole) was added
drop
by-drop with stirring. The mixture was further stirred for 3 hours and
methanol was
removed by evaporation. After filtration with vacuuming, the filtrate was
washed with
distilled water and recrystallized with 95 % ethanol to yield (3, 4-
dihydroxyphenyl)-
13-methyl-tetradecanamine (49.6 g, yield rate 74%).
Example 5
1. Synthesis of 15-methyl-2-hexadecenoic acid
300 ml dry toluene and sodium (138 g, 6.0 mole) were added to a flask. While
stirring, 200 ml anhydrous methanol solution of 13-methyl-tetradecanoate (256
g, 1.0
mole) was added to the flask gradually in 5 minutes. 800 ml more methanol was
added and the mixture refluxed with heating until sodium completely
disappeared. 200
ml water was added and mixture was again refluxed with heating for 30 minutes.
Methanol and toluene were removed by evaporation and water phase was also
removed by extraction. Lipid phase was subject to vacuum fractional
distillation and
140-142°C/l.OmmHg fraction was collected as 13-methyl-1-tetradecanol
(142.6 g,
yield rate 63%).
Chloro-chromic pyridine (161.6 g, 0.75 mole) and 450 ml dichloromethane
were added to a flask. With stirring, 150 ml dichloromethane solution of 13-
methyl-1-
tetradecanol (114 g, 0.50 mole) was slowly added to the flask and stirring
continued
for 1.5 hours at room temperature. Let the mixture stand and separate into two
phases.
Decant upper phase and wash the remainder with 100 ml diethyl ether. Combine
all
liquid and wash sequentially with saturated NaCI, 10% HCI, and saturated NaCI
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solution with 5% NaHC03. Desiccate with anhydrous CaCl2 and remove solvent by
evaporation to yield 13-methyl-tetradecanaldehyde (57.6 g, yield rate 51 %).
13-methyl-tetradecanaldehyde (56.5 g, 0.25 mole), malonic acid (26.5 g, 0.51
mole), and pyridine (30.8 ml, 0.374 mole) were added to a flask and refluxed
with
heating for 3 hours. After cooling down 30% sulfuric acid was added and the
reaction
mixture was stirred for 3 hours under ice bath and then extracted by diethyl
ether.
Diethyl ether was removed by evaporation and product was subject to vacuum
fractional distillation and 170-172°C/l.OmmHg fraction was collected as
15-methyl-2-
hexadecenoic acid (50.9 g, yield rate 76%).
2. Synthesis of 9-methyl-2-decenoic acid
Methyl mono-adipate (102 g, 0.42 mole), isovaleric acid (84.4 g, 0.84 mole),
sodium (0.5 g, 0.022 mole), and 1020 ml methanol were subject to
electrosynthesis at
a direct current of 1.5 A until the reaction mixture was basic. Methanol
evaporated,
the mixture was washed with water and subject to vacuum fractional
distillation.
108°C/23mmHg fraction was collected as 7-methyl-octanoate (53 g, yield
rate 5,2%).
In a similar reaction as in 1 above, 100 ml toluene, sodium (39.5 g, 1.72
mole),
7-methyl-octanoate (52 g, 0.29 mole), and 260 ml anhydrous methanol reacted to
yield
7-methyl-1-octanol (44.3 g, yield rate 85%); 7-methyl-1-octanol (44.3 g, 0.31
mole)
reacted with chloro-chromic pyridine (100 g, 0.465 mole) to yield 7-methyl-
octanaldehyde (31 g, yield rate 70%); and finally, 7-methyl-octanaldehyde (20
g, 0.14
mole), malonic acid (15 g, 0.14 mole), and 15 ml pyridine were added together
and
refluxed with heating for 3 hours. After cooling down 15m130% sulfuric acid
was
added and the reaction mixture was stirred for 3 hours under ice bath and then
extracted by diethyl ether. Diethyl ether was removed by evaporation and
product was
subject to vacuum fractional distillation and 122°C/l.SmmHg fraction
was collected
as 9-methyl-2-decenoic acid (16 g, yield rate 80%).
Example 6
Synthesis of N,N-di-((3-hydroxyethyl)-12-methyl-tridecyl amine
Dichlorosulfoxide (800m1) was added to 13-methyl-tetradecanoic acid (400 g,
CA 02398923 2002-07-30
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1.653 mole) and heated with reflux for 4 hours. To this solution was added
1000 ml
petroleum ether to remove dichlorosulfoxide. After vacuum fractional
distillation,
130-132°C/l.OmmHg fraction was collected as 13-methyl-tetradecanoyl
chloride (375
g, 1.356 mole, yield rate 82.1 %).
Ammonium water (450m1, 28%) was added to a flask and kept under ice bath.
With stirring, 13-methyl-tetradecanoyl chloride (300 g, 1.85 mole) dissolved
in 500 ml
dichloromethane was added drop-by-drop and reacted for 1 hour at room
temperature.
After drying the reaction mixture, extraction by acetyl acetate and
recrystallization,
13-methyl-tetradecanoyl amide (208 g, yield rate 79.5%) was collected.
Sodium (43.9 g, 1.91 mole) was added to anhydrous methanol (2000 ml) in a
flask. After disappearance of sodium, 13-methyl-tetradecanoyl amide (200 g,
0.830
mole) was added and the reaction mixture cooled down to -2°C under ice
salt bath.
With stirring, dry bromine (146.1 g, 0.913 mole) was added drop-by-drop and
stirring
continued for 2.5 hours. Ice bath was removed and reaction mixture heated to
70°C.
10% NaOH (830 ml, 2.04 mole) was added and refluxed for 4 hours. Methanol was
then removed by evaporation and remainder extracted by benzene. After removal
of
benzene by evaporation, vacuum fractional distillation was performed and 160-
162°C/lSmmHg fraction was collected as 12-methyl-tridecyl amine (106 g,
yield rate
60.1 %).
12-methyl-tridecyl amine (106 g, 0.5 mole) was melted at 50°C. With
stirring,
chloroethanol (76.5 g, 1 mole) was added gradually and stirring continued for
half an
hour. Reaction was continued for 8 hours at 95-100°C to completion. The
reaction
mixture was neutralized to pH 7 with 10% NaOH and lipid layer was separated.
Water layer was extracted with benzene for three times and combined with the
separated lipid layer. After removal of benzene and vacuum fractional
distillation,
N,N-di-(~i-hydroxyethyl)-12-methyl-tridecyl amine was collected.
Example 7
Synthesis of 13-methyl-tetradecyl-(2, 4-dihydroxyl) benzene
13-methyl-tetradecanoic acid (36.2 g, 0.15 mole) and anhydrous zinc chloride
(32.7 g, 0.24 mole) were added to a 500 ml flask and heated to 110°C
with oil bath and
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kept for half an hour. While stirring, 1,3-benzenediol (22 g, 0.2 mole) was
added and
reaction heated to 160°C and continued for 2 hours with stirring. After
completion,
reaction was cooled down gradually to 110°C and diluted with 300 ml 15%
HCl (1.04
mole) with stirring and cooled to room temperature and further to below
5°C. After
filtration, the filtrate was washed three times with 5% HCI, and water was
removed.
With vacuum fractional distillation, 230°C/l.OrmnHg fraction was
collected and
decolorized and recrystallized with 80% ethanol to yield 1-(2,4-
dihydroxyphenyl)-1-
(12-methyl)-tridecanone (1 1g, m.p. 87°C, yield rate 87.8%).
1-(2,4-dihydroxyphenyl)-1-(13-methyl)-tetradecanone (20 g, 0.06 mole), Zn-Hg
(200 g, 3.08 mole) and hydrochloric acid (200 ml, 1.67 mole) were added to a
500 ml
flask and refluxed with vigorous stirring for 8 hours and cooled down.
Following
extraction by toluene, removal of toluene and vacuum fractional distillation,
246°C/O.Sg fraction was collected and recrystallized with 95% ethanol
to yield
13-methyl-tetradecyl-(2, 4dihydroxyl) benzene (12 g, m.p. 70°C, yield
rate 62.9%).
Example 8
Synthesis of 13-methyl-tetradecyl-3,4,5-trihydroxybenzoate
To an anhydrous toluene solution (300 ml) of freshly cut sodium (138 g, 6
mole), an anhydrous methanol solution (800 ml) of 13-methyl-tetradecanoate
(256 g, 1
mole) was added gradually with stirring and cooling. After completion of the
addition,
heat with reflux until sodium disappears. Add 400 ml water and reflux for 30
minutes.
Toluene and methanol were removed by evaporation and the lipid layer was
subject to
vacuum fractional distillation. 142-144°C/lmmHg fraction was collected
as 13-
methyl-1-tetradecanol (yield rate 65.0%). 13-methyl-1-tetradecanol (107 g,
0.50 mole)
and 3,4,5-trihydroxybenzoic acid (gallic acid, 94 g, 0.50 mole) were added to
200 ml
toluene and 5.8 ml concentrated sulfuric acid and refluxed with heating. Water
was
removed from the reaction mixture until the temperature of reflux reached
110°C and
toluene was completely removed. The remainder was extracted by ethyl ether and
washed with water. After drying and removal of ethyl ether and
recrystallization with
95% ethanol, 13-methyl-tetradecyl-3,4,5-trihydroxybenzoate (135.4 g, m.p. 64-
66°C,
yield rate 74.0%) was collected.
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The disclosure of Provisional Patent Application No. 601180,677, filed
February 7, 2000, whose date this application seeks benefit of, is hereby
incorporated
by reference.
Obviously, numerous modifications and variations of the present invention are
possible in light of the above teachings. Tt is therefore to be understood
that within the
scope of the appended claims, the invention may be practiced otherwise than as
specifically described herein.
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